Hello, I work at Dynalene Inc., and we have an Aligent HPLC. We have a raw material that contains glycerol and sorbitol and would like to use HPLC to get the concentrations of each for quality control. However, we have run in to a few problems and would like your technical assistance.
Experiment: First, glycerol was held constant at 1% and sorbitol varied in the range of 0-1% with the rest of the solution made the same as the eluent to make a calibration curve for sorbitol (x=weight % sorbitol and y=peak area). Then, sorbitol was held constant at 1% and glycerol varied in the range of 0-1% with the rest of the solution made the same as the eluent to make a glycerol calibration curve. Then, the raw material was diluted 40x to get sorbitol and glycerol in the 0-1% range and the peak areas from that HPLC were used with the calibration curves to get the concentrations of glycerol and sorbitol. This worked out well; however, we want to be able to test the final product which contains 2% of the raw material.
Unfortunately, when I tested the final product (no dilution) the peak areas were much higher than the 40x dilution of the raw material. 40x dilution of the final product still had higher peak areas than 40x dilution of raw material. 100x dilution of the final product still had higher peak area for the sorbitol peak than the 40x dilution of raw material, but lower peak area for glycerol. Still the concentration of glycerol would come out to around 50% (after multiplying by 100 to account for the dilution), when it should be less than 1%. Do you have any ideas as to what is causing this? What should I do to fix it?